IL-10 down-regulates the expression of survival associated gene hspX of Mycobacterium tuberculosis in murine macrophage

w w w . e l s e v ie r . c o m / l o c a t e / b j i d

The

Brazilian

Journal

of

INFECTIOUS

DISEASES

Original

article

IL-10

down-regulates

the

expression

of

survival

associated

gene

hspX

of

Mycobacterium

tuberculosis

in

murine

macrophage

Babban

Jee

_,

_Pawan

_Sharma

_,

_Kiran

_Katoch

_,

_Beenu

_Joshi

_,

_Sudhir

_Kumar

_Awasthi

a_{NationalJALMAInstituteforLeprosyandotherMycobacterialDiseases(ICMR),DepartmentofMicrobiologyandMolecularBiology,} Agra,India

b_{ChhatrapatiShahuJiMaharajUniversity,InstituteofLifeSciences,Kanpur,India}

c_{InternationalCentreforGeneticEngineeringandBiotechnology,ImmunologyGroup,NewDelhi,India} d_{NationalJALMAInstituteforLeprosyandotherMycobacterialDiseases(ICMR),Agra,India}

e_{NationalJALMAInstituteforLeprosyandotherMycobacterialDiseases(ICMR),DepartmentofImmunology,Agra,India}

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Articlehistory:

Received3October2016 Accepted3March2017 Availableonline20April2017

Keywords: Mycobacteriumtuberculosis hspX rmIFN-␥ rmIL-10 Murinemacrophage

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Mycobacteriumtuberculosis(MTB)adoptsaspecialsurvivalstrategytoovercomethekilling mechanism(s)ofhostimmunesystem.Amongstthemanyknownfactors,smallheatshock protein16.3(sHSP16.3)ofMTBencodedbygenehspXhasbeenreportedtobecriticalforthe survivalofMTB.Inthepresentstudy,theeffectofrecombinantmurineinterferon-gamma (rmIFN-␥)andrecombinantmurineinterleukin-10(rmIL-10)ontheexpressionofgenehspX

ofMTBinmurine macrophageRAW264.7hasbeeninvestigated.Byreal-timeRT-PCR,it wasobservedthatthreeincreasingconcentrations(5,25and50ng/ml)ofrmIFN-␥ signif-icantlyup-regulatedtheexpressionofhspXwhereassimilarconcentrationsofrmIL-10(5, 25and50ng/ml)significantlydown-regulatedthehspXexpression.Thiseffectwasnotonly dependentontheconcentrationofthestimulusbutthiswastime-dependentaswell.A contrastingpatternofhspXexpressionwasobservedagainstcombinationsoftwo differ-entconcentrationsofrmIFN-␥andrmIL-10.ThestudyresultssuggestthatrIL-10mediated down-regulationofhspXexpression,inthepresenceoflowconcentrationofrIFN-␥,couldbe usedasanimportantstrategytodecreasethedormancyofMTBinitshostandthusmaking MTBsusceptibletothestandardanti-mycobacterialtherapyusedfortreatingtuberculosis. However,astheseareonlypreliminaryresultsinthemurinecelllinemodel,thishypothesis needstobefirstvalidatedinhumancelllinesandsubsequentlyinanimalmodels mimick-ingthelatentinfectionusingclinicalisolatesofMTBbeforeconsideringthedevelopment ofmodifiedregimensforhumans.

©2017SociedadeBrasileiradeInfectologia.PublishedbyElsevierEditoraLtda.Thisisan openaccessarticleundertheCCBY-NC-NDlicense(http://creativecommons.org/licenses/ by-nc-nd/4.0/).

∗ _{Correspondingauthor.}

E-mailaddress:[email protected](S.K.Awasthi).

http://dx.doi.org/10.1016/j.bjid.2017.03.009

1413-8670/©2017SociedadeBrasileiradeInfectologia.PublishedbyElsevierEditoraLtda.ThisisanopenaccessarticleundertheCC BY-NC-NDlicense(http://creativecommons.org/licenses/by-nc-nd/4.0/).

Introduction

SurvivalofMycobacterium tuberculosis (MTB)in humanhost isthroughbatteryofcomplexmechanismsthatprotectthe pathogenfromhost’skillingresponse.1_{Amongsttheseveral}

factorsknowntobeassociatedwithsurvivalofMTB,small heatshockprotein16.3(sHSP16.3)hasbeenfound indispen-sibleforthesurvivalofMTB.2_{Aninvitrostudysuggeststhat}

thisproteinactsasamolecularchaperonepreventing ther-malaggregationandformsaspecificoligomericstructure.The chaperoneactivityofsHSP16.3istemperaturedependentand ATPindependent.3,4

sHSP16.3wasinitiallyidentifiedasa14kDa immunodomi-nantantigen5,6_{butsubsequentlyitwasfoundthatthisprotein}

accumulatesingreater quantityduringstationary-phaseof MTB,2 _{however, its turnover rate was found low.}7 _{It is a}

majormembraneprotein.8_{Functionalcharacterization}

stud-iesrevealedthatthisproteiniscomposedof144aminoacid residueswithapparentmolecularmassof16,277andshowed amarkedhomologywithamemberof␣-crystallinfamilyor ␣-heatshockprotein(␣-HSP)superfamily.9 _{Itisencodedby}

genehspX(acr,Rv2031c).10_{Inadditiontoplayingavitalrole}

asoneofrescuersduringdenaturationofoneofitsbuilding blocks(i.e.protein),sHSP16.3hasalsobeenfoundtobevital forMTB’svirulence11_{;cellwallthickening,}12_{andmaintenance}

oflong-termdormancy.2,7_{Inaknockoutstudy,itwasfound}

thatdeletionofhspXgenespeedsupthegrowthofMTBboth inmouseandmacrophagemodels.13

TheimportanceofsHSP16.3inthesurvivalofMTBhasbeen wellestablished.However,nopublisheddataareavailableon inhibitionofsynthesisofthisvitalproteinbysuppressingthe expressionofitsencodinggenehspX.Studiescarriedout ear-liershowedthatthepresenceofoxygen,2,14_{reactivenitrogen}

intermediates(RNIs),15_andvitaminC16_increasethe

expres-sionofhspXofMTBinvitro.Themeritofdown-regulationof

hspXexpressionneedstobeexploredinitspossiblerole to preventdormancy,allowmultiplicationoftheparasitesothat anti-mycobacterialagentsusedfortherapyoftuberculosiscan acteffectively,thus,eliminatingtheorganisms.

Cytokinesareadiverseclassofregulatoryproteinsor gly-coproteinsproduced bywhiteblood cells and anumber of other cells inthe body.Cytokines are involved in a broad arrayofbiologicalfunctions. Interferon-gamma(IFN-␥) and interleukin-10(IL-10)whicharerepresentativeofTh1andTh2 cytokines, respectively,are potent immunomodulatorsthat playavitalrole inmodulation oftheimmune responseof macrophageinopposite manner.17 _{IFN-␥isamajor}

activa-torofmacrophage.Besides,itmediatesproductionofeffector moleculessuchasreactivenitrogenintermediates(RNIs)via activation oftumornecrosisfactor-alpha(TNF-␣) cascades, resultinginkillingofintracellularpathogens.18_Ontheother

side,IL-10isakeysuppressorofmacrophagesandexertsits anti-inflammatoryeffectsmainlyonthephagocyticaswellas antigen-presentingcells(APCs).Itprofoundlyinhibitsthe pro-ductionofTh1cytokinesandotherco-stimulatorymolecules aswellasdeactivatestheintracellularanti-bacterial mecha-nisms,helpingtheintracellularpathogenstosurviveinside thehost.19

Thepresentstudyaimedtoinvestigatetheeffectof rmIFN-␥andrmIL-10ontheexpressionofhspXgeneofMTBgrownin murinemacrophageRAW264.7.Afterconductingthereal-time RT-PCRanalysis,thestudyprovidessomeinvitroevidenceof down-regulationofhspXofMTBbyrmIL-10inthepresenceof lowconcentrationofrmIFN-␥.

Materials

and

methods

CultureofMTBandmacrophages

H37Rv strain of MTB (ATCC, USA) was cultured and con-tinuously maintained in Middlebrook 7H9 medium (Difco) supplementedwith10% ADC(BD), 0.2%glyceroland 0.05% Tween-80.MurinemacrophageRAW264.7(ATCC)wascultured inDMEM(Gibco)supplementedwith10%(v/v)heat inactiv-atedFBS(Hyclone),1.46g/ll-glutamine(Sigma),2.3g/lHEPES (Gibco),3.7g/lNaHCO3(Sigma)and10ml/lofPenStrep(Gibco)

andmaintainedat37◦Cina5%CO2humidifiedincubator.

MTBinfectioninmacrophages

AnexponentiallygrowncultureofMTB(OD6000.6)wasused

toinfectthe1.5×106 _{RAW264.7 cellsat1:15multiplicityof}

infection(MOI)in90×100mmCellCultureDish(Corning) con-tainingantibioticfreecompleteDMEM.Singlecellsuspension ofMTBwasprepared.Aftereighthoursofinfection,cellswere washedtwicewithwarmantibioticfreeDMEMand addition-allytreatedwith0.2mg/mlamikacinsulfate(Sigma)fortwo hourstoremoveanyremainingextracellularmicrobes. Sub-sequently,infectedcellswerereplenishedwithfreshcomplete antibioticaddedDMEMandincubatedforthetwotime-points (i.e. 12h and 24h) in the presence or absence of various concentrationsofrmIFN-␥(R&DSystems)andrmIL-10(R&D Systems).

RNAisolation,cDNAsynthesisandreal-timeRT-PCR

Atspecifiedtime-points,MTBinfectedRAW264.7cellswere harvested and total RNA isolation was carried out using GeneJETTM _{RNA purification kit (Fermentas). A total of}

1␮g RNA was transcribed into cDNA by following the protocol of MaximaTM _{first strand cDNA synthesis kit}

(Fermentas). The isolated RNA was treated with RNase-free DNase I (Fermentas). Real-time RT-PCR analysis for the detection of hspX expression was performed using premixed SYBR Green I reaction mixture and following primerspairs(hspX:F:5-CGACAAGGACGTCGACATTA-3,R:5 -CCTTGTCGTAGGTGGCCTTA-3;16SrRNA:F:5 -GCGCAGATAT-CAGGAGGAAC-3,R:5-AAGGAAGGAAACCCACACCT-3)onthe CFX96TouchTM _{Real-TimePCR DetectionSystem (Bio-Rad).}

Theamplificationprocedureinvolvedinitialdenaturationat 94◦Cfor5minfollowedby35cyclesofdenaturationat94◦Cfor 45s,annealingofprimersat65◦Cfor30sandprimer exten-sionat72◦Cfor45s. Aftercompletionofthe 35◦ cycle, the extensionreactionwascontinuedforanother5minat72◦C. 16SrRNAwasusedaninternalcontrolfortheanalysis.

300 200 100 ∗ ∗∗∗ ∗∗∗ ∗∗∗ ∗∗∗ ∗∗∗ hspX

gene expression (in fold)

0 MTB rmlFN-γ (ng/ml) + - 5 12 h 24 h 25 50 + + +

Fig.1–EffectofrmIFN-␥ontheexpressionofgenehspXof MTBinRAW264.7cells.The2−CTmethodwasusedto analyzetherelativechangesinexpressionofhspXin comparisonwith16SrRNA.Thevaluesarethemean±S.D. ofthreeindependentexperiments.*p<0.05,***p<0.0001 betweencontrolandtreatedsamples.

Statisticalanalysis

One-wayANOVA(repeatedmeasures)andBonferroni’spost hoc tests were performed to determine the statistical sig-nificanceamongvariousgroupsusingGraphPadPrism® 5.0 software.p-Valueswereconsideredsignificantifp<0.05.

Results

Thepresentstudywascarriedouttoseewhethertreatment ofIFN-␥and IL-10cytokinesto intracellularMTB,engulfed byRAW264.7cells,modulatestheexpressionofitsgenehspX

encodingsmallheatshockprotein16.3.Forthispurpose,three treatmentgroups were made: (i) rmIFN-␥; (ii)rmIL-10; and (iii)rmIFN-␥and rmIL-10.Two time-points, i.e.12and 24h were usedforeach ofthe abovegroups. Inthe firstgroup, RAW264.7 cells were infectedwithH37Rv strain ofMTBat 1:15MOIfollowingtreatmentwiththreeincreasing concentra-tions(5,25and50ng/ml)ofrmIFN-␥.Thedataobtainedfrom real-timeRT-PCRanalysisshowedthattreatmentof intracel-lularMTBwithaforesaidconcentrationsofrmIFN-␥resulted insignificantup-regulationofhspXexpressionin concentra-tionaswell asinatime-dependentmanner(Fig.1).Inthe secondgroup,thesameconcentrations,i.e.5,25and50ng/ml ofrmIL-10wasusedtotreattheintracellularMTBatthesame specifiedtime-points andthe obtainedfindings were quite different.TreatmentofintracellularMTBwithdifferent con-centrationsofrmIL-10causedsignificantdown-regulationof

hspXexpressioninconcentration-andtime-dependent fash-ion(Fig.2).Inthecombinationgroup(group3)inwhichthe MTBinfectedRAW264.7cellswereincubatedwithcombined dosesofrmIFN-␥andrmIL-10for12and24h,respectively,it wasfoundthatoutoffivecombineddoses,adoseconsisting

0.0 0.5 1.0 1.5 12 h 24 h

∗∗∗

∗∗∗

∗∗∗

∗∗∗

∗∗∗

∗∗∗

gene expression (in fold)

Fig.2–EffectofrmIL-10ontheexpressionofgenehspXof MTBinRAW264.7cells.The2−CTmethodwasusedto analyzetherelativechangesinexpressionofhspXin comparisonwith16SrRNA.Thevaluesarethemean±S.D. ofthreeindependentexperiments.*p<0.05,***p<0.0001 betweencontrolandtreatedsamples.

of5ng/mlofrmIFN-␥and50ng/mlofrmIL-10down-regulates the expressionof hspX at both time-points incomparison withuntreatedcontrolswhereasadoseconsistingof50ng/ml ofrmIFN-␥and5ng/mlofrmIL-10significantlyup-regulates the inductionofhspX intime-dependentmanner. Therest of the three combined doses (rmIFN-␥, 5ng/ml+rmIL-10, 5ng/ml; rmIFN-␥, 25ng/ml+rmIL-10, 25ng/ml; and rmIFN-␥,50ng/ml+rmIL-10,50ng/ml)didnotshowanysignificant effect on the expression ofhspX at boththe experimental time-points(Fig.3).

Discussion

Small heat shock proteins have been identified as vital biomolecules having a substantial role in the pathogene-sis of MTB.2,20 _{MTB has two small heat shock proteins:}

Acr1(␣-crystallinrelatedprotein1/sHSP16.3/HSP16.3/16kDa antigen/HspX2_{)andAcr2/HrpA.}21_{sHSP16.3isoneofthecrucial}

proteinsofMTBthatfacilitatessurvivalofMTBinitshost dur-ingprolongedperiodsofinfection13_{mostprobablybyaltering}

the expression ofcertain microRNAs.22 _{Expressionof hspX}

hasbeenfoundtobemainlyregulatedbyatwo-component responseregulatordosR,dormancysurvivalregulator (previ-ously knownasdevR, Rv3133c).23 _{Thefactors whichtrigger}

theinductionofhspXarenotfullyknown.However,itis sug-gestedthathypoxiaisamajorstimulusbehindtheexpression ofthisgene.2_Besides,NO,15_vitaminC,16_{andingestionofMTB}

bymacrophage11_{havealsobeenreportedtoup-regulatethe}

expressionofhspX.

Cytokines are potent immunomodulators having pro-nounced effectson immunologicresponsesgeneratedbya numberofcelltypesinnormalaswellasinpathologic condi-tions.AlthoughtheroleofIFN-␥andIL-10inthepathogenesis

0 50 100 150 200 12 h 24 h ∗∗∗ ∗∗∗ 50 5 50 25 5 - + + + + + + 5 50 50 25 5 -MTB rmIFN-γ (ng/ml) rmIL-10 (ng/ml) hspX

gene expression (in fold)

Fig.3–EffectofrmIFN-␥andrmIL-10ontheexpressionof genehspXofMTBinRAW264.7cells.The2−CTmethod wasusedtoanalyzetherelativechangesinexpressionof

hspXincomparisonwith16SrRNA.Thevaluesarethe mean±S.D.ofthreeindependentexperiments.*p<0.05, ***p<0.0001betweencontrolandtreatedsamples.

oftuberculosisaswellasintheintracellularsurvivalofMTB24

iswellknown,theireffectontheexpressionofsurvival asso-ciatedgenehspXhasnotbeenpreviouslyreported.

Ourstudy has demonstrated that three increasing con-centrations (5, 25, and 50ng/ml) of rmIFN-␥ significantly up-regulated the expression of hspX whereas similar con-centrations of rmIL-10 (5, 25, and 50ng/ml) significantly down-regulatedthehspXexpression.Thiseffectwasfoundto bedependentonboththeconcentrationofthestimulusand time.AcontrastingpatternofhspXexpressionwasobserved whenMTBinfectedmacrophageswereexposedto combina-tionsoftwodifferentconcentrationsofrmIFN-␥andrmIL-10, i.e.lowexpressionofhspXtoacombinationoflow concentra-tionofrmIFN-␥andhighconcentrationofrmIL-10versushigh expressionofhspXtoacombinationofhighconcentrationof rmIFN-␥andlowconcentrationofrmIL-10.Themechanisms ofsuch cytokine mediated modulation ofhspX expression areyettobefullyexplored,whichmaybemediatedthrough dormancyregulondosR.Anearlierstudyhasreportedthat tar-geteddisruptionofthisregulonresultsintheeliminationof

hspXexpression.23

Growthretardationisacharacteristicfeatureof mycobac-terial dormancy.25 _{Studies carried out earlier with mutant}

strains showed that over-expression of hspX gene slows thegrowth ofbothMTBand Mycobacteriumsmegmatis2 _and

probably pushesthe bacteria to enter dormant state.13 _As hspX appears to play a central role in dormancy of MTB andpotentanti-tuberculosisdrugsdonoteffectivelyacton dormantorganisms,26_{rIL-10drivendown-regulationofhspX}

expression,inthepresenceoflowconcentrationofrIFN-␥, wouldpossiblydecreasethechancesofdevelopingdormancy andincreasetheintracellulargrowthofMTB.Thus,itwould beabetteropportunitytoanti-tuberculosisdrugstoacton

multiplying MTB. Such intervention could also be of help inprevention/eliminationofpersistersandinshorteningof therapy.Thismaybetoooptimistictorelyonlyononegene orbiomarkerlikehspX asanumberofgenesare knownto beassociatedwithsurvivalofMTBinsidethehost.1,27–29_At

this moment, this isessentially ahypothesis which needs tobevalidatedfirstinhumancelllinesandsubsequentlyin experimentalanimalmodelsmimickingthelatentinfection usingclinicalisolatesofMTB.Ifsuccessful,thismaythenbe validated in humansubjects byfollowing properapproved andwellplannedprotocols.

Conclusion

This study demonstrates that rmIL-10 down-regulates the expression of gene hspX of MTB in murine macrophage. Furtherstudiesinvolvingotherexperimentalmodelsare warr-antedtovalidatethefindingsofthisstudy.

Conflicts

of

interest

Theauthorsdeclarenoconflictsofinterest.

Acknowledgments

ThefinancialsupportfromDepartmentofScienceand Tech-nology,NewDelhiintheformofProjectNo.SR/SO/HS-66/2007 isdulyacknowledged.AuthorsarehighlythankfultoDirector, International Center for Genetic Engineering and Biotech-nology,NewDelhiforprovidingessentialresearchfacilities; ArvindPandey,Director,NationalInstituteofMedical Statis-tics, NewDelhi forstatisticalanalysis; SantoshKumarand ZavedSiddiqui,InternationalCenterforGeneticEngineering andBiotechnology,NewDelhifortheirtechnicalsupport.

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